The subject of the invention is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one purified natural or synthetic polypeptide, specific to the epidermis, having a role in intercorneocyte cohesion. The subject of the invention is also a cosmetic or pharmaceutical composition comprising a mixture of polypeptides derived from the proteolysis of the purified polypeptide, a method of cosmetic treatment for strengthening intercorneocyte cohesion and a method of cosmetic treatment for reducing intercorneocyte cohesion, and therefore for promoting desquamation.
Human skin consists of two compartments, namely a deep compartment, the dermis, and a superficial compartment, the epidermis.
The dermis provides the epidermis with a solid support. It is also its feeder component. It consists mainly of fibroblasts and an extracellular matrix itself mainly composed of collagen, elastin and a substance called ground substance, these components being synthesized by the fibroblast. Leukocytes, mastocytes or tissue macrophages are also found therein. It also consists of blood vessels and of nerve fibers.
Natural human epidermis is composed mainly of three types of cells which are the keratinocytes, which are highly predominant, the melanocytes and the Langerhans"" cells. Each of these cell types contributes, through its specific functions, to the essential role played by the skin in the organism.
The epidermis is conventionally divided into a basal layer of keratinocytes which constitutes the germinative layer of the epidermis, a so-called prickle cell layer consisting of several layers of polyhedral cells arranged on the germinative cells, a so-called granular layer consisting of flattened cells containing distinct cytoplasmic inclusions, the keratohyalin granules, and finally a top layer called horny layer (or stratum corneum), consisting of keratinocytes at the final stage of their differentiation, called corneocytes. These are anucleated, mummified cells which are derived from the keratinocytes.
The corneocytes are mainly composed of a fibrous matrix containing cytokeratins, surrounded by a very resistant structure 15 nm thick, called horny or hornified envelope. The stacking of these corneocytes constitutes the horny layer which is responsible for the barrier function of the epidermis. During the normal desquamation process, the most superficial corneocytes become detached from the surface of the epidermis.
Intercellular structures derived from the desmosomes, called corneosomes or corneodesmosomes, have been described in the horny layer. Recent studies have shown their key importance in intercorneocyte cohesion as well as in the desquamation process. In particular, a close correlation exists between cell dissociation and proteolysis of certain corneodesmosomal components such as desmoglein I.
Several serine proteases of the trypsin or chymotrypsin type appear to be involved in the proteolysis of the corneodesmosomes, in particular the chymotryptic enzyme of the horny layer (stratum corneum chymotryptic enzyme).
Numerous pathological conditions of the skin are characterized by the production of a thick horny layer and by an abnormal desquamation, that is to say by hyperkeratosis. The latter may occur on any anatomical skin area and in a wide variety of clinical contexts. Its physiopathological substratum and its cause are varied.
By way of example, there may be mentioned:
xerosis (or dryness of the skin),
ichthyoses,
psoriasis,
certain benign or malignant tumour lesions,
reactive hyperkeratoses.
Other pathological conditions are characterized by transdifferentiation or metaplasia, at the level of the mucosae, Malpighian or otherwise, but normally nonhornified, which become hornified, that is to say which become covered with an abnormal epithelium, producing a horny layer at its surface. Although the genital mucosae and those of the upper aerodigestive tracts are most often involved, these metaplasias may be seated in various anatomical areas.
By way of examples, there may be mentioned
leukokeratosis of the uterine neck during prolapsus,
buccal leukokeratoses,
keratotic benign tumour lesions of the Malpighian mucosae
By contrast, some pathological manifestations cause thinning of the epidermis and in particular of the horny layer, resulting in excessive fragility of the skin covering. It may be seated in various anatomical areas, its cause is variable and it may be constitutional or acquired.
By way of examples, there may be mentioned:
trophic skin disorders of the lower limbs in patients carrying vascular pathological conditions: varicose veins, arteriopathies (diabetes, arteriosclerosis and the like),
trophic skin disorders in the context of an algodystrophic syndrome,
trophic disorders following abnormal cicatrization.
The purification and knowledge of the polypeptides involved in intercorneocyte cohesion is one of the routes which could allow the production of products for combating the effects of an excess or a deficiency of polypeptides of this type, in particular at the surface of the skin.
One of the objects of the invention is to provide a composition comprising a polypeptide involved in intercorneocyte cohesion in purified form.
After long and laborious studies because of its low representation among the proteins of the epidermis, and its high instability and its high sensitivity to proteases, the applicant has identified, isolated and purified by biochemical techniques, from a human epidermis, a polypeptide specific to the hornified epithelia. This polypeptide, which will also be called elsewhere in the text xe2x80x9ccorneodesmosinexe2x80x9d is expressed in the horny layer of the epidermis and is involved in intercorneocyte cohesion. The applicant has determined the primary amino acid sequence thereof.
The subject of the invention is therefore a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one purified natural or synthetic polypeptide, the said polypeptide being characterized in that it corresponds to the following amino acid sequence SEQ ID NO: 1:
The polypeptide of the invention may be of natural or synthetic origin. Synthetic is understood here to mean any polypeptide obtained chemically or by production in an organism after introducing into this organism the components necessary for this production.
The polypeptide of the invention may be derived from any possible origin, namely either animal, in particular mammalian and still more particularly human, origin or plant origin or from microorganisms (inter alia viruses, phages or bacteria) or from fungi, without prejudging the fact that it is present naturally or otherwise in the said organism of origin.
Preferably, the polypeptide of the invention is of natural origin, purified from mammalian tissues, particularly from mammalian skin.
Preferably, the polypeptide of the invention is purified from human skin and still more preferably from human epidermis.
As indicated above, intercorneocyte cohesion is apparently due, inter alia, to the existence, in the horny layer, of polypeptides specific to the structures involved in the intercorneocyte junction.
Accordingly, the polypeptide of the invention is specific to the horny layer and to the granular layer and, preferably, the polypeptide according to the invention is specific to the structures involved in the intercorneocyte junction, particularly of the corneodesmosomes.
It is known, in general, that the mature polypeptides which are found in cells are derived from the maturation of precursors which contain, in their sequence, the sequence of the mature polypeptide.
Accordingly, the invention also relates to a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, any polypeptide whose sequence partly consists of the sequence of the polypeptide of the invention.
It is also known that the polypeptides may undergo post-translational modifications such as the formation of disulphide bonds, specific proteolytic cleavages, the addition of carbohydrates (glycosylation), phosphorylation, in particular at the level of the serines and/or of the threonines and/or of the tyrosines, and/or combination with lipids.
The invention therefore relates to a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one polypeptide of the invention which has undergone post-translational modifications or not.
The polypeptide of the invention may have undergone one or more post-translational modifications.
Preferably, the polypeptide according to the invention is glycosylated and/or phosphorylated.
It is known to classify polypeptides according to their isoelectric point. Preferably, the polypeptide of the invention is basic.
Of course the primary amino acid sequence as well as the various post-translational modifications undergone by the polypeptide are responsible for the fact that the said polypeptide may be characterized by its molecular weight, expressed in kilodaltons.
The polypeptide of the invention has an apparent molecular weight, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), of between 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons.
Most preferably, the polypeptide of the invention is a basic, phosphorylated, glycosylated polypeptide having an apparent molecular weight of between 50 and 60, preferably between 52 and 56 kilodaltons.
Thus preferably, the subject of the invention is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one polypeptide of the invention, the said polypeptide being phosphorylated, basic and glycosylated and having an apparent molecular weight of between 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons.
It is also known that the primary amino acid sequence of a polypeptide determines sites specifically recognized by proteases which, once the recognition of these sites has been achieved, will, with or without attachment to the said polypeptide, induce its cleavage by proteolysis.
Accordingly, the invention also relates to a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one mixture of polypeptides derived from the proteolysis of the polypeptide of the invention.
As described above, some pathological conditions may be due to excessive desquamation which may be assumed to be due to a deficiency of polypeptides involved in intercorneocyte cohesion.
Some pathological manifestations cause thinning of the epidermis and, in particular, of the horny layer, resulting in excessive fragility of the skin covering, which may be seated in any anatomical skin area and may have various causes, constitutional or acquired.
Accordingly, the composition according to the invention is intended for treating the thinning of the epidermis, and in particular the horny layer, and/or for treating excessive fragility of the skin covering and/or for strengthening intercorneocyte cohesion and/or inducing the thickening of the horny layer.
Likewise, the composition according to the invention is intended for treating the thinning of the epidermis, and in particular of the horny layer and/or for treating excessive fragility of the skin covering and/or for strengthening intercorneocyte cohesion and/or inducing the thickening of the horny layer.
Likewise, the composition of the invention may be used for causing localized thickening of the horny layer at the level of the skin regions which have to be subjected to repeated microtraumas.
By way of example, there may be mentioned the preventive treatment of subepidermal blisters (xe2x80x9campullaexe2x80x9d) in sportspeople.
The subject of the invention is also a method of cosmetic treatment for treating the thinning of the epidermis, and in particular of the horny layer, and/or for treating excessive fragility of the skin covering and/or for strengthening intercorneocyte cohesion and/or inducing the thickening of the horny layer, characterized in that a cosmetic composition according to the invention is applied to the skin of the subject to be treated.
The subject of the invention is also a method of cosmetic treatment for treating trophic skin disorders, for example of patients carrying vascular pathological conditions such as varicose veins or arteriopathies (diabetes, arteriosclerosis and the like), trophic skin disorders of patients carrying algodystrophic syndromes or those following cicatrization disorders.
Analysis of the primary amino acid sequence of the protein according to the invention shows that it has recognition and binding sites for known proteases or specific sites for cleavage by chemical agents. There may be mentioned, as example, the sites for Chymotrypsin, Cathepsin, proteinase K, Subtilisin, protease V8, Thermolysin, Thrombin, Trypsin, Papain, Pepsin, Proline-Endopeptidase, Endoproteinase GluC, Endoproteinase LysC, Endoproteinase AspN, Endoproteinase ArgC (Clostripain), Myxobacter AL117, Elastase, chymotryptic enzyme of the horny layer, cyanogen bromide, N-chlorosuccinimide or specific sites for acid hydrolysis (70% formic acid, 7 M Guanidine-HCl, 400xc2x0 C., 24 h).
Intercorneocyte cohesion is apparently due to the existence, in the horny layer, of polypeptides soecific to the structures involved in the intercorneocyte junction such as, in particular, the polypeptide of the invention. It has been seen that certain hyperkeratotic pathological conditions could be linked to an excessive intercorneocyte cohesion, due in particular to the polypeptide of the invention.
Accordingly, the subject of the invention is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, an effective quantity of at least one protease active on the polypeptide of the invention, chosen from Endoproteinase LysC, Endoproteinase GluC, Proline-Endopeptidase, Thrombin, Pepsin, Myxobacter AL117 and Elastase.
These proteases may be isolated from plants, animals, bacteria, viruses or fungi.
The polypeptide of the invention may be glycolysed. Accordingly, the composition as defined above may contain, in addition, a glycosidase which may be chosen from those isolated from plants, animals, fungi or microorganisms, in particular bacteria. There may be mentioned, by way of example, neuraminidases, mannosidases, galactosidases, glucosidases, N-acetylglucosaminidases and N-acetylgalactos aminidases.
As seen above, some pathological conditions are characterized by the production of a thickened epidermal horny layer and by abnormal desquamation, that is to say by hyperkeratosis which may occur in any anatomical skin area, in various clinical contexts and whose physiopathological substratum and cause may be varied.
The invention also relates to a cosmetic or pharmaceutical composition comprising a protease, as defined above, for treating, for example, hyperkeratosis, xerosis (or dryness of the skin), ichthyoses, psoriasis, certain benign or malignant hyperkeratotic tumour lesions, and reactive keratoses.
The invention also relates to a cosmetic or pharmaceutical composition comprising a protease, as defined above, for treating pathological conditions which are characterized by transdifferentiation or metaplasia, at the level of mucosae, Malpighian or otherwise, but normally nonhornified, which become hornified such as, for example, leukokeratosis of the uterine neck during prolapsus, buccal leukokeratoses, or benign or malignant hyperkeratotic tumour lesions of the Malpighian mucosae.
Another object of the invention is to provide a method of cosmetic treatment for combating excessive intercorneocyte cohesion and therefore for increasing desquamation and in particular the excesses due to the polypeptide of the invention, which method consists in applying to the skin a cosmetic composition comprising at least one protease having a specific recognition and/or binding and cleavage site within the primary amino acid sequence of the polypeptide of the invention.
It is known that a protein is synthesized in the cells from a template of deoxyribonucleic acids encoding the said protein. It is also known that the genetic code is degenerate. Accordingly, the amino acid sequence of the polypeptide of the invention may be derived from various natural or synthetic deoxyribonucleic acid sequences. Synthetic deoxyribonucleic acid sequence is understood here to mean any sequence obtained chemically or by genetic engineering.
The said deoxyribonucleic acid sequences may be derived from any possible origin, namely either animal, in particular mammalian and still more particularly human origin or plant origin or from microorganisms (inter alia viruses, phages, or bacteria) or from fungi, without prejudging the fact that they are present naturally or otherwise in the said organism of origin.
The applicant has isolated, purified and sequenced, using molecular biology techniques, in particular the screening of libraries for expression of complementary deoxyribonucleic acids prepared from human epidermis, a deoxyribonucleic acid fragment encoding the polypeptide of the invention.
The subject of the invention is also therefore a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, any deoxyribonucleic acid sequences, natural or synthetic, all or part of which encodes the primary amino acid sequence of the polypeptide of the invention.
During these studies, the applicant has been able to isolate and purify a deoxyribonucleic acid sequence encoding the primary amino acid sequence of the polypeptide of the invention from human skin.
Particularly, the subject of the invention is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, an isolated deoxyribonucleic acid fragment comprising at least the following coding nucleotide sequence SEQ ID NO: 2:
The subject of the invention is also a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, a sense or antisense ribonucleic acid sequence corresponding to the said sequence SEQ ID NO: 2.
Its subject is also the use of the said deoxyribonucleic acid sequences for the production of the polypeptide of the invention or of a corresponding ribonucleic acid by any known technique such as for example synthesis in vitro, from reconstituted media, or synthesis by organisms.
The subject of the invention is also the use of the polypeptide of the invention or of its proteolysis fragments, and of any synthetic peptide deduced from its sequence, for preparing or purifying, optionally from the epidermis, any molecule, structural or functional, capable of binding specifically to the said purified polypeptide or to the said purified proteolysis fragments or to the said synthetic peptide. This molecule may in particular correspond to other structural proteins specific to the corneodesmosomes and various enzymes of the horny layer, of the xe2x80x9cproteasexe2x80x9d, xe2x80x9cglycosidasexe2x80x9d or xe2x80x9cphosphatasexe2x80x9d type.
The subject of the invention is also the use of the polypeptide of the invention or of its proteolytic fragments and of any synthetic peptide deduced from its sequence, for preparing specific monoclonal antibodies and antisera, intended in particular for purifying this protein and its fragments. By extension, the subject of the invention is also any use of the said sequence for producing recombinant antibodies or antibody fragments, regardless of the biological system used for producing the latter.
Whatever their nature, the compositions of the invention may be ingested, injected or applied to the skin (over any skin area of the body) or the mucosae (buccal, jugal, gingival, genital, conjunctival and the like).
Preferably, the compositions of the invention are applied to the skin or the mucosae.
Depending on the mode of administration, the compositions according to the invention may be provided in any of the galenical forms normally used.
For a topical application to the skin, the composition may take the form, in particular, of an aqueous or oily solution or of a dispersion of the lotion or serum type, of emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or conversely (W/O), or of suspensions or emulsions of soft consistency of the aqueous or anhydrous cream or gel type, or of microcapsules or microparticles, or of vesicular dispersions of the ionic and/or nonionic type or of foams or alternatively in the form of aerosol compositions also comprising a pressurized propellant. These compositions are prepared according to the customary methods.
For injection, the composition may be provided in the form of an aqueous or oily lotion or in the form of a serum. For the eyes, it may be provided in the form of drops, and for ingestion, it may be provided in the form of capsules, granules, syrups or tablets.
The quantities of the various constituents of the compositions according to the invention are those conventionally used in the fields considered.
These compositions constitute in particular cleansing, protective, treatment or care creams for the face, for the hands, for the feet, for the large anatomical folds or for the body, (for example day creams, night creams, make-up removing creams, foundation creams, antisun creams), fluid foundations, make-up removing milks, protective or care body milks, antisun milks, skincare lotions, gels or foams, such as cleansing lotions, artisun lotions, artificial tanning lotions, bath compositions, deodorant compositions containing a bactericidal agent, aftershave gels or lotions, depilatory creams, compositions against insect bites, antipain compositions or compositions for treating certain skin diseases such as eczema, rosacea, psoriasis, lichens and severe pruritus.
The compositions according to the invention may also consist of solid preparations constituting cleansing soaps or cakes.
The compositions may also be packaged in the form of an aerosol composition also containing a pressurized propellant.
The composition according to the invention may also be a composition for the care of the scalp, especially a shampoo, a hair setting lotion, a treatment lotion, a hair styling cream or gel, a dyeing (especially oxidation dyeing) composition optionally in the form of dyeing shampoos, restructuring lotions for the hair, a permanent-waving composition (especially a composition for the first stage of a permanent waving), a lotion or gel against hair loss, an antiparasitic shampoo and the like.
The composition may also be for dentibuccal use, for example a toothpaste. In this case, the composition may contain customary adjuvants and additives for compositions for buccal use and especially surfactants, thickening agents, humectants, polishing agents such as silica, various active ingredients such as fluorides, in particular sodium fluoride, and optionally sweetening agents such as sodium saccharinate.
When the composition is an emulsion, the proportion of fatty phase may range from 5% to 80% by weight, and preferably from 5% to 50% by weight relative to the total weight of the composition. The oils, waxes, emulsifiers and coemulsifiers used in the composition in the form of an emulsion are chosen from those conventionally used in the cosmetic field. The emulsifier and the coemulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5 to 20% by weight relative to the total weight of the composition. The emulsion may, in addition, contain lipid vesicles.
When the composition is an oily gel or a solution, the fatty phase may represent more than 90% of the total weight of the composition.
In a known manner, the cosmetic composition may also contain adjuvants common in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, screening agents, odour absorbers and colouring materials. The quantities of these various adjuvants are those conventionally used in the cosmetic field, and for example from 0.01% to 10% of the total weight of the composition. These adjuvants, depending on their nature, may be introduced into the fatty phase, into the aqueous phase and/or into the lipid spherules.
As oils or waxes which can be used in the invention, there may be mentioned mineral oils (petroleum jelly), vegetable oils (liquid fraction of shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils (Purcellin oil), silicone oils or waxes (cyclomethicone) and fluorinated oils (perfluoropolyethers), beeswaxes, carnauba or paraffin waxes. Fatty alcohols and fatty acids (stearic acid) may be added to these oils.
As emulsifiers which can be used in the invention, there may be mentioned for example glycerol stearate, polysorbate 60 and the PEG-6/PEG-32/Glycol Stearate mixture sold under the name TefoseR 63 by the company Gattefosse.
As solvents which can be used in the invention, there may be mentioned the lower alcohols, especially ethanol and isopropanol, and propylene glycol.
As hydrophilic gelling agents which can be used in the invention, there may be mentioned the carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylate/alkylacrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays, and, as lipophilic gelling agents; there may be mentioned modified clays such as bentones, metal salts of fatty acids such as aluminium stearates and hydrophobic silica, ethylcellulose and polyethylene.
The composition may contain other hydrophilic active agents, such as proteins or protein hydrolysates, amino acids, polyols, urea, allantoin, sugars and sugar derivatives, water-soluble vitamins, plant extracts and hydroxy acids.
As lipophilic active agents, there may be used retinol (vitamin A) and its derivatives, tocopherol (vitamin E) and its derivatives, essential fatty acids, ceramides, essential oils, salicylic acid and its derivatives.
According to the invention, the composition may combine at least one extract of at least one Iridaceae with other active agents intended especially for the prevention and/or treatment of skin conditions. Among these active agents, there may be mentioned, by way of example:
agents reducing skin differentiation and/or proliferation and/or pigmentation such as retinoic acid and its isomers, retinol and its esters, vitamin D and its derivatives, oestrogens such as oestradiol, kojic acid or hydroquinone;
antibacterials such as clindamycin phosphate, erythromycin or antibiotics of the tetracycline class;
antiparasitic agents, in particular metronidazole, crotamiton or pyrethroids;
antifungal agents, in particular the compounds belonging to the imidazole class such as econazole, ketoconazole or miconazole or their salts, the polyene compounds, such as amphotericin B, the compounds of the allylamine family, such as terbinafine, or octopirox;
antiviral agents such as acyclovir;
steroidal anti-inflammatory agents such as hydrocortisone, betamethasone valerate or clobetasol propionate, or nonsteroidal anti-inflammatory agents such as, for example, ibuprofen and its salts, diclofenac and its salts, acetylsalicylic acid, acetaminophen or glycyrrhizic acid;
anaesthetic agents such as lidocaine hydrochloride and its derivatives;
antipruritic agents such as thenaldine, trimeprazine or cyproheptadine;
keratolytic agents such as xcex1- and xcex2-hydroxycarboxylic or xcex2-ketocarboxylic acids, their salts, amides or esters and more particularly hydroxy acids such as glycolic acid, lactic acid, salicylic acid, citric acid and, in general, fruit acids and 5-n-octanoylsalicylic acid;
anti-free-radical agents, such as xcex1-tocopherol or its esters, superoxide dismutases, certain metal chelators or ascorbic acid and its esters;
antiseborrhoeic agents such as progesterone;
antidandruff agents such as octopirox or zinc pyrithione;
anti-acne agents such as retinoic acid or benzoyl peroxide.
Accordingly, according to a specific embodiment, the composition according to the invention also comprises at least one agent chosen from antibacterial, antiparasitic, antifungal, antiviral, anti-inflammatory, antipruritic, anaesthetic, keratolytic, anti-free-radical, antiseborrhoeic, antidandruff and anti-acne agents, and/or agents reducing skin differentiation and/or proliferation and/or pigmentation.